Engineer bifunctional antibacterial enzymes for treatment of S. aureus infections

摘要：抗生素耐药性是金黄色葡萄球菌(金黄色葡萄球菌)感染的主要原因。一个完整的 三分之二的医院相关金黄色葡萄球菌感染和约50%的社区获得者现在是 耐甲氧西林(MRSA)。耐甲氧西林金黄色葡萄球菌每年在美国造成45万人感染，它对 所有死亡中有一半是由耐药细菌造成的。金黄色葡萄球菌多重耐药的高发生率 而其他细菌强调了对能够对抗这些危险的下一代抗生素的需求 病原体。一种越来越引人注目的治疗策略利用重组酶，例如 模拟葡萄球菌溶葡萄球菌酶(LST)，它能降解细胞壁肽聚糖，引起细菌溶解和 死亡。LST是一种高效的抗葡萄球菌药物，对药物敏感和 金黄色葡萄球菌的抗药性菌株。虽然LST在对抗危险的金黄色葡萄球菌方面具有巨大的潜力 作为一种系统管理的治疗方法，它的效用受到特定限制的限制。首先，作为一个 LST是一种26,942道尔顿的小蛋白，可通过肾滤过迅速从血液中清除。在诊所里，这个 事实可能需要频繁、高剂量的注射才能实现细菌的完全清除。为您提供以下选项 使用更低的剂量和更少的频率将降低成本，减轻患者负担，并改善 治疗结果。其次，由于金黄色葡萄球菌的突变，LST容易产生抗药性 FEMA基因，它改变了酶所针对的特定的肽聚糖键。协同式2-剂 治疗已被证明可以降低LST耐药性的风险。我们在这里建议构造一个模块 基于与免疫球蛋白Fc结构域融合的双功能溶素平台。FC域是自然而然的 双价展示支架，我们的目标是利用经过验证的旋钮和孔双特定FC工程策略 创建异双功能Fc-裂解酶嵌合体。具体地说，我们将融合LST催化和细胞壁结合 将结构域连接到一个异二聚体旋钮和孔Fc的一个链上，我们将融合Sa2原噬菌体内肽酶 域连接到第二个链。两条Fc链的异源配对将产生单个分子实体 它整合了两个互补的细胞壁水解酶，已知可以发挥抗S。金星协同效应。两种功能 Fc-Lysin对金黄色葡萄球菌细胞壁的双管齐下攻击应该会将获得性耐药性降至最低。此外，功能界别 结构域将通过(I)增加分子的大小来延长双功能溶素的循环半衰期 超过肾脏首次通过肾小球滤过的限制，以及(Ii)接触新生儿Fc受体 (FcRN)，它积极回收免疫球蛋白抗体，并促进其异常长的半衰期。总体来说，这 该项目寻求开发一个模块化平台，用于设计高性能抗菌酶， 利用分子内协同作用杀死耐药细菌病原体。

Abstract: Antibiotic resistance complicates the majority of Staphylococcus aureus (S. aureus) infections. A full two thirds of hospital-associated S. aureus infections and ~50% of those acquired in the community are now methicillin-resistant (MRSA). MRSA causes >450,000 infections in the US each year, and it is responsible for half of all deaths caused by drug-resistant bacteria. The high incidence of multi-drug resistance in S. aureus and other bacteria underscores the need for next-generation antibiotics capable of combating these dangerous pathogens. An increasingly compelling therapeutic strategy leverages recombinant enzymes, such as Staphylococcus simulans lysostaphin (LST), which degrade cell wall peptidoglycan causing bacterial lysis and death. LST is a highly potent anti-staphylococcal agent with proven efficacy against both drug-sensitive and drug-resistant strains of S. aureus. While LST holds great potential for combatting dangerous S. aureus infections, its utility as a systemically administered treatment is constrained by specific limitations. First, as a small protein of 26,942 daltons, LST is rapidly cleared from the blood stream by renal filtration. In the clinic, this fact might necessitate frequent, high dosing to achieve complete bacterial clearance. Providing the option to use lower doses and less frequent administration would reduce costs, ease patient burden, and improve treatment outcomes. Second, LST is subject to development of S. aureus resistance by virtue of mutations in the femA gene, which alters the specific peptidoglycan bond targeted by the enzyme. Synergistic 2-agent treatments have been shown to mitigate the risk of LST resistance. We propose here to construct a modular bifunctional lysin platform based on fusions with immunoglobulin Fc domains. The Fc domain is a natural bivalent display scaffold, and we aim to leverage validated knob and hole bispecific Fc engineering strategies to create heterobifunctional Fc-lysin chimeras. Specifically, we will fuse the LST catalytic and cell wall binding domains to one chain of a heterodimeric knob and hole Fc, and we will fuse the SA2 prophage endopeptidase domain to the second chain. The heterologous pairing of the two Fc chains will create a single molecular entity that integrates two complementary cell wall hydrolases known to exert anti-S. aureus synergy. The bifunctional Fc-lysin's two pronged attack on S. aureus cell walls should minimize acquired resistance. Additionally, the Fc domain will serve to extend the bifunctional lysin's circulation half-life by (i) increasing the molecule's size beyond the limit for first-pass glomerular filtration in the kidney, and (ii) engaging the neonatal Fc receptor (FcRn), which actively recycles IgG antibodies and promotes their exceptionally long half-lives. As a whole, this project seeks to develop a modular platform for engineering high performance antibacterial enzymes that capitalize on intramolecular synergy to kill drug-resistant bacterial pathogens.